Project 6: Mothes/Blanchard Conformational trajectories of inhibitor-bound HIV-1 envelope Summary The design of effective antiviral therapies directed against HIV Env pursued by this program requires a fast method to determine the underlying molecular mechanism of HIV Env inhibition by small molecule inhibitors. Towards this end, we have established single-molecule Fluorescence Resonance Energy Transfer (smFRET) methods to visualize directly the structural dynamics of HIV Env in the context of the complete trimer on the surface of HIV virions. As a proof of concept we have successfully applied smFRET to the X4-tropic HIV-1 NL4-3- Our work reveals that the unliganded HIV Env is conformationally dynamic and intrinsically capable of sampling receptor CD4- and 17b-stabilized conformations. Importantly, the co-receptor binding site is more likely to be accessed from the CD4-induced state. Binding of CD4 lowers the energy of the CD4-stabilized conformation and prepares the HIV Env for co-receptor binding. These data explain how HIV Env is activated in a stepwise manner by CD4 and the co-receptor. Here, we will extend the application of smFRET to include physiologically relevant R5-tropic HIV-1 Env isolates. Dually modified HIV-1 Env variants will be used to determine the conformational trajectories of HIV Env induced by CD4 mimetics and antagonists. This will allow distinction between CD4 mimics such as NBD556 that induce CD4-like conformational changes and antagonists such as DMJ-l-228. smFRET imaging will also permit an understanding of conformational molecular events underlying HlV-1 inactivation either by prematurely activating Env, kinetic traps or allosterically diverting Env into off-pathways. As such, these smFRET technologies will provide a critical tool to determine the underlying molecular mechanism of HIV Env antagonism and inactivation developed by the Program Project Team and aid in a rational design of antiviral therapies that exploit the intrinsic vulnerabilities of HIV. smFRET technologies will accelerate pharmaceutical discovery by enabling screening for desired conformational effects that block, trap or inactivate HIV-1 Env.